Biomedical subjects
B A Gaëta
Publications and source records attributed to B A Gaëta.
Biocomputing education by the Australian National Genomic Information Service.
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The 5'-flanking region of the alpha 2MR/LRP gene contains an enhancer-like cluster of Sp1 binding sites.
We have sequenced a genomic DNA fragment containing the promoter and 5'-flanking region of the alpha 2MR/LRP. A cluster of five Sp1 sites situated over 600 base pairs away from the putative transcription start site doubles the activity of the promoter. A similar increase in activity was observed when this region was replaced by the SV40 enhancer, but the presence of both the cluster of Sp1 sites and SV40 enhancer gave no more transcription than either region alone. Within the previously described promoter region we have shown that only the most proximal Sp1 binding site influences transcription in CHO cells. The Sp 1 site situated 346 bp upstream of the putative transcription start site and previously described DNAse protection footprints had no effect on promoter activity in CHO cells. We also describe an NRF-1 binding site situated 143 bp upstream of the putative transcription start site. Deletion of the central 4 bp of this site caused a 60% decrease in transcription. No sterol regulatory (SRE-1) sites, used in the LDL receptor promoter for control of expression by cholesterol, were found in the alpha 2MR/LRP 5'-flanking region. However, one SRE-1 site was identified in the 5'-untranslated region of alpha 2MR/LRP.
Saturation mutagenesis of the Drosophila tRNA(Arg) gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation.
Transcription of eukaryotic tRNA genes is dependent on the A- and B-Box internal control regions (ICRs) and the upstream transcription modulatory region. The B-Box ICR spans nucleotides 52 to 62 and directs the primary binding of transcription factor C as the first step in the formation of a transcription complex. The conservation of the sequence of the B-Box in all tRNA species reflects its importance in both the expression of the gene and the processing, structure and function of the gene product. In order to identify the nucleotides essential to the promoter function of the B-Box ICR, site-directed mutagenesis was used to generate all the possible single point mutations at positions 52 to 58, 61 and 62 of a Drosophila melanogaster tRNA(Arg) gene. The effect of these mutations on gene transcription was evaluated using in vitro transcription and template exclusion competition assays. Optimal activity was displayed by the wild type tDNA(Arg) B-Box sequence but several other sequences supported in vitro transcription at wild type levels. The majority of mutants, however, showed lower efficiency in the in vitro transcription assay. Of the single point mutations, those at positions 53, 55, and 56 had a critical effect on gene function in Drosophila and HeLa transcription extracts and transcription factor interaction most likely requires base contacts at these positions. Since the effect of several of the point mutations cannot be explained in terms of possible major or minor groove contributions the possibility is raised that local DNA geometry also is an important determinant in specifying B-Box function.